Alteration of cell membrane with B7

ABSTRACT

Methods and compositions are provided for the persistent modification of cell membranes with exogenous proteins so as to alter the function of the cell to achieve effects similar to those of gene therapy, without the introduction of exogenous DNA. DNA sequences, the proteins and polypeptides embodying these sequences are disclosed for modulating the immune system. The modulations include down-regulation, up-regulation and apoptosis.

This application is a U.S. National Stage filing under 35 U.S.C. 371from International Application No. PCT/US01/20946 filed Jul. 2, 2001 andpublished in English as WO 02/02751 A2 on Jan. 10, 2002, which claimedpriority from U.S. Provisional Application No. 60/215,580 filed Jun. 30,2000, which applications and publication are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to the persistent modification of cell membranesso as to alter the function of the cells. The compositions and methodsof this invention achieve effects similar to those of gene therapywithout the introduction of exogenous DNA. A useful alteration of cellfunction is the induction of apoptosis.

BACKGROUND OF THE INVENTION

It has long been the goal of experimental biology and medicine to inducecells to behave in predictable ways and to alter the behavior of cellsin ways that are beneficial to a subject. For example, if undesiredcells could be induced to alter their behavior to undergo apoptosiswhile normal cells retain normal function, subjects with a diseasecaused by proliferation of undesired cells would obtain relief from thedisease. Similarly, if tissue-rejecting cells can be eliminated or theirbehavior changed, transplantation with tissues foreign to the subjectcan be successful.

Gene therapy has been proposed for selected diseases in order to corrector modify pathological or physiological processes. In gene therapy as itis generally termed, specific DNA is introduced into a tissue and organ,where it is produces various proteins that will correct or amelioratethe condition. It is an unpredictable therapy, depending on potentiallydangerous expression vectors and the uncertain efficiency of delivery,which is often low. Moreover, gene therapy is considered to havedangerous side effects, such as sustained expression in desired cells ortissues past the desired duration of therapy, or the introduction ofgenetic modifications in undesired tissues or cells. Because of suchadverse effects, as discovered in human clinical trials, much caution isadvised before gene therapy is put in practice.

DNA-based gene therapy has been the subject of intense studies duringthe past few decades because of the tremendous potential it offers forthe treatment of inherited diseases and other pathologic conditions forwhich the expression of selected proteins may offer treatment. Forexample, gene therapy can be used for immunomodulation either to enhancethe capacity of the immune response to deal with infections or tumors,or to down-regulate the immune response for the prevention ofautoimmunity or foreign graft rejection. Despite vast efforts in thepast two decades, the safe and effective application of gene therapy tothe treatment of diseases has been extremely limited. Among the apparentdrawbacks of gene therapy are the possibility of causing permanentchange in the DNA complement of the host; uncertain tissue specificity;expression of the encoded protein beyond the intended duration oftherapy; and high cost. It would therefore be extremely beneficial ifcells and tissues could be modified to express proteins of interest in ashort period of time without the introduction of foreign DNA.

Whereas gene therapy has generally focused on the problem of deliveringnucleic acids into cells, much fundamental knowledge concerning theimportant role of cell surface molecules has been gained through studiesof signal transduction in cells of the immune response, which arereadily accessible and have well-understood functions. The immuneresponse is regulated by the interaction of several different celltypes, which react to the presence of foreign antigens. The adaptiveimmune response is critical to the survival of vertebrates in anenvironment full of pathogenic microorganisms. Individuals who, due toinborn genetic defects, exposure to chemotherapeutic agents or infectionby such viruses as human immunodeficiency virus (HIV), lack a fullyfunctional immune response are susceptible to infections that anindividual with a healthy immune response would readily withstand.However, the immune system does not always function in ways that arebeneficial to the organism. Its dysregulation leads to autoimmunity andtumors. The immune system also serves as a barrier to thetransplantation of foreign grafts, such as cells taken from anindividual other than the transplant recipient. Transplantation permitsthe replacement of failed cells, tissues, or organs in otherwiseterminal diseases, while bone marrow transplantation can treathematopoietic disorders, malignancies, autoimmune disorders, and otherdiseases. For transplantation to be successful, it is necessary eitherto suppress the adaptive immunity or to “teach” the recipient's immunesystem to accept these foreign antigens as native.

The immune response to foreign antigens is initiated by naive T cellsthat use clonally-expressed T cell receptors (TCRs) to recognizeantigens such as peptides presented by self-major histocompatibilitycomplex (MHC) molecules. This recognition reaction, when accompanied bycostimulatory signals provided by antigen-presenting cells (APCs), hasbeen thought to result in full T cell activation. A productive T-cellresponse is now seen as requiring three distinct signals. Signal 1 isgenerated by T-cell receptor interaction with the majorhistocompatibility complex (MHC) antigen/peptide on antigen-presentingcells (APCs). Signal 2 is mediated by the engagements of costimulatorymolecules, such as B7/CD28 and CD40/CD40L, on T cells and APCs. Signal 3is transduced via cytokines elaborated by T cells and APCs that havereceived both Signal 1 and 2. The transduction of these 3 signals drivesT cells and APCs to proliferation and differentiation into effectors forthe generation of a productive immune response. The lack of any of thesesignals during the T-cell response results in T-cell anergy and immunenonresponsiveness. For example, tumors evade the immune system bypreventing the transduction of one of these signals.

Upon activation, T cells proliferate and differentiate into effectorcells that evoke immunological mechanisms responsible for the clearanceof antigens from the system. A period of death then follows during whichmost of the activated T cells undergo apoptosis-mediated“activation-induced cell death” (AICD) and effector activity subsides.Apoptosis is a complex process that involves a series of extra- andintracellular signals that converge on the activation of enzymes calledcaspases that commit the cell to apoptosis.

Transplantation of foreign cells (such as bone marrow and stem cells),tissues (such as pancreatic islets), and organs (such as kidneys,hearts, livers) has become an important and effective therapeuticalternative for patients with selected terminal diseases. Thetransplantation of foreign grafts between genetically different patients(allografts between members of the same species or xenografts betweenmembers of different species) is, however, limited by the ability tocontrol the immunological recognition and rejection of the graft by therecipient.

Bone marrow (BM) transplantation has been viewed as an extraordinarilypromising treatment for hematopoietic and autoimmune disorders and forcertain cancers. One obstacle to bone marrow transplantation is thepossibility of rejection of the transplanted tissue, mediated by thehost's T cells and NK cells. Graft-versus-host-disease (GvHD) is anotherpossible adverse consequence of bone marrow transplantation. Donor Tcells in the transplanted tissue can mount an immune response againstthe host's vital organs, often leading to death of the host.Host-versus-graft reactions and GvHD therefore limit the clinical use ofbone marrow transplantation, which might otherwise be widely used totreat various diseases and to prevent foreign graft rejection.

Pharmacological agents that cause immunosuppression are now a mainstayof regimens for the control of allograft rejection. Although such drugsare effective in reducing the severity of rejection episodes, they arenonspecific and fail to create a state of permanent graft-specifictolerance. Continuous exposure of the recipient to theseimmunosuppressive agents is therefore associated with a significantlyincreased risk of opportunistic infections and malignancies. The needremains to develop more selective and long-lasting methods to prevent BMrejection.

Additionally, these nonspecific immunosuppressive agents can induceserious and undesirable side effects in the host. These adverse effectsoften outweigh the benefits for patients with diseases in which the bodyidentifies certain parts of itself as “foreign” and launches an adaptiveimmune attack that results in autoimmunity, such as is observed in TypeI diabetes, arthritis, lupus, and multiple sclerosis. It would be verydesirable to be able to “teach” the immune system to tolerate the“foreign” self-antigen.

It would also be very desirable to be able to “teach” the immune systemto rid the organism of tumor cells. T cell-mediated cellular immunity isthe most critical acquired response against tumors. A series ofexperimental studies has provided evidence that tumors evadeT-cell-mediated immunity by several different mechanisms. Thesemechanisms include: i) lack of Signal 1, due to inefficient display ofMHC/tumor antigen bimolecular complexes on tumor cells or defects in thetransduction of this signal; ii) absence of Signal 2, due to the absenceof costimulatory molecules on tumor cells; iii) induction of anergy in Tcells; and iv) physical elimination of effector T cells via apoptosis.Although all of these mechanisms may be operative in patients with alarge tumor burden, the lack of costimulation is believed to play themost critical role.

The need therefore remains to develop more rapid, selective andlong-lasting methods to modulate cell function without the introductionof nucleic acids into cells for therapeutic purposes. The need alsoremains to develop a means of accomplishing the end of gene therapywithout many of the risks attendant to the introduction of exogenousnucleic acids into an organism. Since much cell function is controlledthrough the transduction of signals at the cell surface, a generallyapplicable method of attaching an agent to a surface would be useful.Among the uses of such a method would be: the modulation of cellfunction without the introduction of nucleic acids into the cell; theaccomplishment of the end of gene therapy by alternative and potentiallypreferable means; and the manipulation of an organism's immune responsein order to diminish that response, as to treat autoimmunity or toforestall graft-versus-host disease, or to increase that response, as totreat tumors or infections.

SUMMARY OF THE INVENTION

The present invention provides a method of modifying a surface, themethod comprising: (1) contacting a surface with one member of a bindingpair which binds to the surface to form a decorated surface; andsubsequently (2) contacting the decorated surface with a compositioncomprising the second member of the binding pair operably linked to anagent capable of modifying the surface and the function thereof. Thebinding pair may be avidin/biotin, streptavidin/biotin orantigen/antibody. By “avidin” is meant avidin and any fragment orderivative of avidin which retains strong binding to biotin. By“streptavidin” is meant streptavidin and any fragment or derivation ofstreptavidin which retains strong binding to biotin. Conversely, by“biotin” is meant any fragment or derivative of biotin which retainsstrong binding to avidin and streptavidin.

When the surface is a cell surface, the agent may be any compoundinducing apoptosis; any agent down-regulating the immune system; anyagent up-regulating the immune system; any adhesion molecule for celltracking; any growth factor; or an antibody ligated to a toxin such asricin or phytotoxin. When the surface is a cell surface, the contact ofstep 1 produces a “decorated cell.”

When the agent is selected to induce apoptosis, the agent may be adeath-inducing molecule such as FasL, mFasL, TRAIL, TNF-a, TWEAK and thelike. When the agent is selected to down-regulate the immune system, theagent may be an anti-inflammatory cytokine such as IL-4, IL-10, TGF-βand the like. When the agent is selected to up-regulate the immunesystem, the agent may be a costimulatory molecule such as B7, CD40L andthe like or a pro-inflammatory cytokine such as IL-2, IL-12, IL-15,lymphotactin and chemokine.

In a preferred embodiment, the first member of the binding pair isbiotin, the second member of the binding pair is a chimeric proteincomprising streptavidin and a death-inducing molecule and the surfacecomprises target cells, tissues, organs or tumors.

In a more preferred embodiment, the first member of the binding pair isSulfo-NHS-LC-biotin, the second member of the binding pair is SA-mFasLand the surface comprises a cell displaying the Fas receptor. Inconstructing the second member of the binding pair, streptavidin isfused to the extracellular portion of FasL through recombinant genetechnology and the chimeric protein SA-mFasL is produced.

This construct is used in a method to modulate the immune system whereina subject to be treated is first administered biotin to form decoratedcells, followed by administration of SA-mFasL to form chimeric decoratedcells. Only those cells in the tissue, organ or tumor which arebiotinylated and express Fas will undergo apoptosis. In general, thesecells include those cells which cause immuno-mediated injuries.

In another aspect of the invention, a subject who will benefit fromdown-regulation of the immune system is first administered biotin toform decorated cells and is then administered a construct comprisingavidin or streptavidin and a down-regulating molecule. Subjects who willbenefit from down-regulation of the immune system include subjectssuffering from autoimmunity, graft-versus-host disease, allergies,septic shock and vascular diseases. Subjects undergoing tissue or organgrafts also benefit from the down-regulation of the immune system.

In another aspect of the invention, a subject who will benefit fromup-regulation of the immune system is first administered biotin to formdecorated cells and is then administered a construct comprising avidinor streptavidin and an up-regulating molecule. Subjects who will benefitfrom an up-regulation of the immune system include subjects sufferingfrom neoplasia or infectious disease.

In another aspect of the invention, systemic effects are avoided bybiotinylating the cells ex vivo by isolation of the target or selectionof a target that can be treated topically, followed by reintroduction ofthe decorated cells into the subject with subsequent second contact.

In another aspect of the invention, the members of the binding pair forma covalent bond with each other. In a preferred aspect, the first memberof a binding pair comprises a cysteine residue and the second member ofthe binding pair comprises a cysteine residue. The two members are firstplaced together under conditions disfavoring the formation of adisulfide bond between the first member of the binding pair and thesecond member of the binding pair. In a subsequent step, the two membersare placed under conditions favoring the formation of a disulfide bondbetween the first member of the pair and the second member of the pair.

In another aspect of the invention, the first member of a binding paircontacts a cell surface, a virus, the surface of a glass particle, thesurface of a polysaccharide particle or the surface of a plasticparticle to form a decorated surface. The decorated surface is thencontacted with the second member of the binding pair, to form a seconddecorated surface. The cell, virus or particle comprising the seconddecorated surface is contacted with a cell or cell component of anorganism either in vitro or in vivo, for the purpose of therapy,diagnosis, cell sorting or identification.

A recombinant nucleic acid is provided comprising the extracellularportion of FasL fused with streptavidin (SA-mFasL). Gene product isprovided which is then attached to the cell surface, previouslybiotinylated in vivo. Methods are taught for the use of gene productsfor long-lasting elimination of Fas expressing cells.

The methods of this invention can be modified to cause persistentbinding of any protein to targeted cells, to mimic the result of DNAgene therapy without modification of the cell genetic material. Thispersistent binding of a protein to a targeted cell can be considered tobe gene therapy at the protein level.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the method to modify cellmembrane for the expression of exogenous proteins for new functions.

FIG. 2 shows the restriction fragment analysis of the expression vectorcontaining the indicated clones.

FIG. 3 shows biotin expression in vivo.

FIG. 4 shows the in vitro expression of SA-mFasL on the surface ofsplenocytes.

FIG. 5 shows SA-mFasL expression on the surface of bone marrow cells.

FIG. 6 shows donor chimerism in lethally irradiated animals.

FIG. 7 shows a graphic summary of allogeneic cells decorated withSA-mFasL blocking in vivo alloreactive responses.

FIG. 8 shows a pancreatic islet cell expressing FasL (red) andstreptavidin (B) compared to a control islet (A).

FIG. 9 shows T cells modified to express SA-mFasL, activated by ConA.

FIG. 10 shows expression of SA-CD40L on splenocytes.

DETAILED DESCRIPTION OF THE INVENTION

Because of the noted problems associated with gene therapy as it iscommonly used, that is, the introduction and integration of foreign DNAinto the genome, a method was sought for the accomplishing the resultsof gene therapy, that is, long lasting modification of cell function,without the limitations of DNA introduction. The question was raised asto whether the benefits of gene therapy could be obtained by thedelivery of exogenous proteins, rather than nucleic acids, to the cells.A method that would safely permit modification of the cell membrane toachieve transient or long-lasting display of therapeutic molecules couldhave wide-spread application in the clinic. As disclosed in the presentapplication, Applicant has discovered and here discloses such a method,which has the added advantage of rapid cell surface expression.

Because of the thermodynamically favored association of avidin orstreptavidin with biotin, the complex formed can modify cell propertiesfor a period that can rival or surpass the duration of the effectelicited by gene therapy that requires the introduction of nucleicacids. The present invention teaches a method of attaching an agent to asurface. Such an agent can be, by way of example and not of limitation,any of a series of immunomodulatory molecules such as death ligands,costimulatory molecules and cytokines. It is useful to attach suchagents to surfaces, especially the cell surface, since such agents canbe used to physically or functionally eliminate autoreactive,alloreactive, xenoreactive and inflammatory cells to prevent allograftand xenograft rejection, graft-versus-host disease and to treatautoimmunity, septic shock, inflammation, allergies, infections,neoplasias, and vascular diseases. Furthermore, this method can be usedto display proteins with defined function on the surface of cells for invivo trafficking as for the homing of selected hematopoietic cells fortherapeutic purposes.

Definitions:

For the purposes of the present application, the following terms havethese definitions:

“Agent” means a composition which elicits a measurable biologicalresponse or which is useful in the diagnosis, prevention or treatment ofa disease or in the sorting or identification of cells, cell componentsor viruses.

“Binding pair” means two compositions that readily bind to each other;that have affinity for one another; or whose association isthermodynamically and kinetically favored.

“Cell surface” has its normal meaning in the art, comprising thephospholipid bilayer of a cell membranes and the molecules directly orindirectly associated with the bilayer.

“Decorated surface” means a surface to which a member of a binding pairhas become bound.

“Expression” means, in addition to its conventional meaning in the art,the placement of a functional protein on or within a cell.

“lastic” means polystyrene, polypropylene, polyethylene or any otherpolymer capable of being formed into a solid surface.

“Protein” means a protein or polypeptide that is native, non-native,synthetic or modified as by covalent binding.

“Surface” means a cell surface, the surface of a virus, the surface of aparticle (e.g., the surface of a glass particle, the surface of apolysaccharide particle, the surface of a plastic particle), the surfaceof a phospholipid bilayer or the surface of a solid matrix.

“Target cell” means a cell targeted for apoptosis, that is, a cell inwhich it is desired to induce apoptosis.

An important application of regulation of the immune response is theinduction of tolerance to alloantigens, xenoantigens and autoantigens toprevent foreign graft rejection, treat or prevent autoimmunity and GVHD.T cells are the most critical cells of adaptive immunity, whichrequires, three distinct signals (Signal 1, 2 and 3) for a productiveresponse. Signal 1 is generated by T-cell receptor interaction with theMHC/peptide complex on APCs. Signal 2 is mediated by the engagements ofcostimulatory molecules, such as B7/CD28 and CD40/CD40L on T cells andAPCs. Signal 3 is transduced via cytokines elaborated by T cells andAPCs that have received both Signal 1 and 2. The lack of any of thesesignals during T-cell response to antigens may serve as one of the mosteffective mechanisms by which tolerance is induced. Upon activation, Tcells undergo a state of antigen-driven proliferation that allows up toa 1200-fold clonal expansion. A period of death then ensues during whichmore than 95% of the activated T cells undergo apoptosis programmed celldeath) while the remaining cells differentiate into memory cells as theamount of antigen in the system declines.

Apoptosis or “programmed cell death” plays a central role in both thedevelopment and homeostasis of multicellular organisms. Apoptosis can beinduced by multiple independent signaling pathways that converge upon afinal effector mechanism consisting of multiple interactions betweenseveral “death receptors” and their ligands, which belong to the tumornecrosis factor (TNF) receptor/ligand superfamily. The bestcharacterized death receptors are CD95 (“Fas”), TNFR1 (p55), deathreceptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). A finaleffector mechanism is mediated by the caspase group of proteins.

In copending International Application PCT.US01/02256, the teachings ofwhich are incorporated by reference, it is disclosed thatFas-/FasL-induced apoptosis plays a central role in protectingimmunologically privileged sites, such as the central nervous system,testes and eyes from immune attack. Allogeneic and xenogeneic tissuestransplanted into these sites, for instance, are resistant to rejection.Of great importance is the major role apoptosis plays in maintaininghomeostasis in the immune system via activation-induced cell death(AICD). AICD is primarily mediated by apoptopic signals transduced bythe Fas/FasL interaction. Fas is a 45 kDa, type I, cell surface proteinconsisting of a cysteine-rich extracellular domain, which transduces adeath signal. Fas mediates effector function by interacting with FasL, atype II membrane protein of about 40 kDa. T cells are the primary celltype in the body that express FasL, upon activation. The expression ofthis molecule makes the activated T cells susceptible to apoptosis,which is induced in an autocrine fashion as Fas engages with FasL on thesame cell. Fas on an activated T cell may also interact in a paracrinefashion with FasL on another activated cell. These two pathways ofapoptopic T cell death mediated by the Fas/FasL system serve as ahomeostatic mechanism for controlling the size of antigen-stimulated Tcell clones. Deregulation of this system results in immunologicaldisorders.

The molecular mechanism of Fas/FasL-mediated apoptosis has been studiedin great detail. Binding of three FasL molecules with Fas inducestrimerization of Fas receptor via c-terminus death domains (dds), whichin turn recruit an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FADD/caspase-8 results in proteolytic cleavage of proenzymecaspase-8 into active caspase-8 that, in turn, initiates the apoptosisprocess by activating other downstream caspases through proteolysis,including caspase-3.

Apoptosis of activated T cells results in tolerance to allografts andxenografts, including bone marrow and other organ and cellulartransplantation. Purging of activated T cells also relieves the symptomsof allergies and other immune-induced diseases. Included in the latterare auto-immune disorders such as multiple sclerosis, lupuserythematosus, sarcoidosis and rheumatoid arthritis. Many disorders,including some tumors, are dependent on lymphocyte functions that leadto persistence of the disorder. Many hematological disorders could betreated with bone marrow stem cell transplants if the immune responsecould be regulated so as to induce tolerance to the foreign stem cells.Among these disorders are leukemias, lymphomas, aplastic anemia, sicklecell and Cooley's anemia and the like. All of these disorders may becontrolled permanently or temporarily by apoptosis of activated immunecells, including T cells.

Strategies are provided for immunomodulation using rapid cell-surfacedisplay of proteins, which comprise the construction of chimeric cDNAsencoding the functional portions of an immunoregulatory protein with onemember of a binding pair. Table 1 is a summary of such constructs.Choice of constructs may be based on factors such as: the nature of theforeign antigen provoking adaptive immunity; whether the relief to besought is temporary or permanent; whether a commitment to death isdesired or additional downstream regulation of apoptosis is preferred.It is to be understood that the constructs listed below arerepresentative of the invention only and are not limitations. Forexample, the contructs have been made using core streptavidin (CSA).

Fragments and derivatives of streptavidin as well as the entirestreptavidin molecule can be as readily used to make equivalentconstructs. Therefore, in the specification, SA is generally used tomean whole, fragmented or derivatized streptavidin. Those skilled in theart can readily, without undue experimentation, follow the teachings ofthis invention to make constructs comprising a first member of a bindingpair and a second member of a binding pair ligated to a knownimmunomodulatory molecule.

TABLE 1 SEQ Vector Insert ID # Function Application pCSA- 6His-RS-CSA- 1Apoptosis Induce apoptosis in immune- mFasL SRIPE-Extracellular reactivecells, smooth muscle portion of FasL cells for the treatment ofimmune-based disorders (GVHD, rejection of foreign grafts, autoimmunity,inflammation, allergies, septic shock and likes) and vascular diseases.pCSA- 6His-RS-CSA- 7 Apoptosis Induce apoptosis in immune- TNFαSRIPE-Extracellular reactive cells, smooth muscle portion of TNFα cellsfor the treatment of immune-based disorders (GVHD, rejection of foreigngrafts, autoimmunity, inflammation, allergies, septic shock and likes)and vascular diseases. pIL-4- Mature portion of IL- Immune Down-regulateimmune- CSA 4-6His-RS-CSA- down- reactive cells for the SRIPE regulationtreatment of immune-based disorders (GVHD, rejection of foreign grafts,autoimmunity, inflammation, and likes). pIL-10- Mature portion of IL- 5Immune Down-regulate immune- CSA 10-6His-RS-CSA- down- reactive cellsfor the SRIPE regulation treatment of immune-based disorders (GVHD,rejection of foreign grafts, autoimmunity, inflammation, and likes).pTGFβ- Active portion of 3 Immune Down-regulate immune- CSATGF-β-6His-RS- down- reactive cells for the CSA-SRIPE regulationtreatment of immune-based disorders (GVHD, rejection of foreign grafts,autoimmunity, inflammation, allergies, septic shock, and likes). pCyto-Active portion of Immune Down-regulate immune- CSA other down- reactivecells for the immunoregulatory regulation treatment of immune-basedcytokines-6His-RS- disorders (GVHD, rejection CSA-SRIPE of foreigngrafts, autoimmunity, inflammation, allergies, septic shock, and likes).pIL-2- Mature portion of IL- 4 Immune up- Up-regulate immune-reactiveCSA 2-6His-RS-CSA- regulation cells for the treatment of SRIPEinfections and cancers. pB7-CSA Extracellular portion 2 Immune up-Up-regulate immune-reactive of B7-6His-RS-CSA- regulation cells for thetreatment of SRIPE infections and cancers. pCSA- 6His-RS-CSA- 6 Immuneup- Up-regulate immune-reactive CD40L SRIPE -Extracellular regulationcells for the treatment of portion of CD40L infections and cancers.padhesion- Active portion of Immune Down-regulate immune- CSA adhesionmolecules - down- or reactive cells for the 6His-RS-CSA- upregulationtreatment of immune-based SRIPE disorders (GVHD, rejection of foreigngrafts, autoimmunity, inflammation, allergies, septic shock, and likes).Up-regulate immune-reactive cells for the treatment of infections andcancers. pchemo- Active portion of Immune Down-regulate immune-kines-CSA immunoregulatory down- or reactive cells for thechemokines-6His- upregulation treatment of immune-based RS-CSA-SRIPEdisorders (GVHD, rejection of foreign grafts, autoimmunity,inflammation, allergies, septic shock, and likes). Up-regulateimmune-reactive cells for the treatment of infections and cancers.

A preferred production cell for the production of chimeric compositionsencoded by the DNA constructs is the Drosophila system that iscommercially available. However, those skilled in the art of producingchimeric compositions will recognize that many other expression systemsand vectors are suitable for production of the chimeric compositions ofthis invention. Included in these other systems are Escherichia coli,yeast and mammalian cell cultures.

The inventions disclosed in copending international applicationPCT/US00/34554, the teachings of which are hereby incorporated byreference, are directed to the apoptosis of lymphocytes and tumor cells.The present invention discloses an improved method of expressingimmunomodulatory molecules with therapeutic interest on the surface ofcells via binding pair coupling. Avidin or streptavidinibiotin is anexample of a binding pair useful in the present invention. A modifiedFasL protein is described in detail as an example of such a composition.Bone marrow cells, endothelial cells, splenocytes, tissues such aspancreatic islets and organs such as hearts are examples of cellsamenable to cell surface alteration. It has now been found that thismethod can be used to down-regulate or up-regulate immune responses toantigens. This method, therefore can be used for the prevention offoreign graft rejection, for the prevention or treatment of autoimmunityand the up-regulation of immune response to tumors and infections.Additionally, it is provided that proteins other than those withimmunoregulatory functions can be bound to cells without causingsystemic side effects.

The invention will now be described with specific examples. Theexperimental procedures described herein are representative ofcompositions and methods for persistent binding “expression” of agentsto cells, tissues or organs. One embodiment specifically described is achimeric composition, SA-mFasL, produced by fusion of DNA coding forcore streptavidin protein with that of DNA coding for FasL andexpressing the chimeric composition (SEQ#1) in a suitable productioncell.

The target cells are first biotinylated ex vivo or in vivo and thenSA-mFasL is administered in order to cause apoptosis, resulting in, forexample, induction of donor-specific long-term survival and/ortolerance. Another example that is described in detail is thetransplantation of pancreatic islet cells along with immune cellstreated according to the methods of this invention, with subsequentenduring pancreatic insulin production. Another example is the long termsurvival of ectopic heart transplants. Others examples are alsoprovided. Following the teachings of the following examples, one skilledin the art can readily apply these to alter the membrane function ofother cells.

Alternatively, the DNA encoding such chimeric compositions may beapplied ex vivo or in vivo, leading to permanent changes such as theapoptosis of activated T cells.

The following examples are disclosed in order to illustrate the presentinvention as it is applied in practice and do not limit the scope of theappended claims.

EXAMPLE 1

Cloning of Core Streptavidin for the Generation of Chimeric Proteins.

The present invention discloses technology for cell-surfacemodifications to express exogenous proteins without the introduction ofnucleic acids into cells and comprises: i) generation of a chimericmolecule consisting of core streptavidin and functional domains of adesired protein, ii) modification of the cell membrane with biotin, andiii) decoration of the biotinylated cells with the chimeric molecule(FIG. 1). To accomplish this, genomic DNA encoding streptavidin wascloned from S. avidinii using specific primers in PCR. 5′ and 3′ primerswere designed to incorporate sequences for selected restriction enzymesites and amino acids that allow three dimensional flexibility, properfolding and function. The gene was cloned into the TA cloning vector,sequenced and subcloned into the pMT/Bip/V5-HisA vector for expressionin a high-yield insect expression system (DES™, Invitrogen).

EXAMPLE 2

Construction of Chimeric Genes for Expression in Production Cells.

Total RNA was prepared from human cell lines or peripheral blood cellsand 2 ng of this RNA was reverse transcribed into the first strand ofDNA using oligo (dT) 18 as a primer for reverse transcriptase. One-tenthof this cDNA preparation was then amplified, using three sets of senseand antisense primers specific for human IL-2, IL-4, IL-10, TGF-β, FasL,TNF-α, B7.1, and CD40L in 8 separate PCR amplifications. The 5′ and 3′primers were designed to include restriction enzyme sites for cloningand several amino acid residues to facilitate the proper folding of theproduct. These primers amplified DNA bands of expected sizes for allthese genes of interest. These PCR products were then cloned into the TAcloning vector (Invitrogen, San Diego, Calif.) and a library preparedfrom this material was screened using the same oligonucleotide primersin PCR amplifications. The positive clones were digested withappropriate restriction enzymes, leading to the release of expected sizeof fragment for each gene (FIG. 2) shown in Table 1. All indicatedclones were sequenced and found to have the expected characteristics.These clones were then fused to CSA in the pMT/Bip/V5-HisA vector eitheras N-terminus or C-terminus proteins to facilitate correct threedimensional structure and function (see SEQ IDs in Table 1).

Chimeric proteins were subcloned in frame with the Drosophila BiPsecretion signal in the pMT/BipNV5-HisA vector for expression in ahigh-yield insect expression system (DES™, Invitrogen). Recombinantvectors were transiently transfected into Drosophila S2 cells usingcalcium phosphate. Cells were then pulsed with copper sulfate toactivate the inducible metallothionein promoter driving the expressionof chimeric proteins. Culture medium was collected at various timespost-activation, dialyzed to remove the copper sulfate, and analyzed forchimeric proteins using ELISA and Western blot.

EXAMPLE 3

Biotinylation of Cells, Expression of Recombinant Proteins, and TimeKinetics of Expression in Vitro

The therapeutic use of this protein-based approach requires successfulbiotinylation of cells, tissues, or organs under physiologicalconditions and attachment to these cells chimeric proteins consisting ofCSA and molecules with therapeutic potential (FIG. 1).

The optimum conditions for biotinylation were first determined. Singlecell suspensions were prepared from spleen or bone marrow of the rat.One million cells were incubated in various concentrations, ranging from1.5 to 150 μM, of EZ-Link biotin (trade name of Sulfo-NHS-LC-biotin,Pierce, Rockford, Ill.) in saline at room temperature for 30 minutes.After extensive washing to remove free biotin, cells were eithercultured or used for staining with fluorescein (APC)-labeledstreptavidin in order to assess the level of biotinylation. 100% of thecells were positive for biotin at 5 μM biotin concentration (data notshown).

To determine how long biotin persists on actively dividing cells and theoptimum dose of biotin (15 μM), biotinylated splenocytes were culturedin the presence of a T cell mitogen, concanavalin A (ConA), to stimulateT-cell proliferation. ConA-stimulated and unstimulated cells wereharvested at various times post culture and stained withAPC-streptavidin and analyzed by flow cytometry. Splenocytes withoutstreptavidin and splenocytes with biotin alone served as backgroundstaining. Over 50% of the cells maintained biotin on the surface for 20days, the longest time point tested. A significant portion (>35%) ofactively dividing cells also expressed biotin at this time period. Itwas next tested to determine whether biotinylation interferes with theproliferation of splenocytes. Splenocytes were activated with 2.5 μg/mlConA for various days in 96-titer plates. Cultures were pulsed withtritiated thymidine, harvested, and analyzed for DNA-associatedradioactivity as an indication of proliferation. Biotinylatedsplenocytes proliferated in response to ConA activation with similarkinetics and levels as compared with unmanipulated splenocytes,suggesting that biotinylation at concentrations ranging between 1.5–15μM does not have a significant effect on the proliferative function ofthe cells (data not shown).

Biotinylated vascular endothelial cells in culture were tested todetermine the time kinetics of biotin on the cell surface. The rationalefor this set of experiments is to test how long biotin persists on thesurface of differentiated cells with minimal turnover in tissues such asheart vasculature. Monolayer cultures of rat aortic endothelial cellswere treated with 15 μM concentration of biotin in six-well plates for30 min at room temperature. Cells were extensively washed and one wellof the culture was digested with trypsin at different times postbiotinylation, single cell suspension was prepared and analyzed in flowcytometry using APC-streptavidin. A significant portion of endothelialcells (>15%) expressed biotin 20 days in culture, the longest time pointtested.

Splenocytes were tested for the expression of one of the recombinantproteins with immunomodulatory function, the extracellular portion ofFasL fused with core streptavidin (SA-mFasL). Splenocytes werebiotinylated as stated above, incubated with 100 ng SA-mFasL for 20 min,and maintained in a 37° C. incubator for defined periods of time atwhich cells were stained with an anti-FasL antibody. Almost all thecells expressed SA-mFasL on day 5 and significant number of cells (>25%)expressed SA-mFasL on day 10, the last time point analyzed (FIG. 4).

One of the objectives of the proposed protein-based gene therapy to useimmunomodulatory molecules such as SA-mFasL to facilitate theengraftment of BM cells in a foreign environment. Therefore, BM cellswere tested for biotinylation and expression of FasL at the proteinlevel. BM cells were harvested from femurs and tibia of rats usingstandard protocols. Single cells suspension was prepared and thenbiotinylated under the above conditions. After extensive washing toremove biotin, BM cells were treated with SA-mFasL (50 ng/million cells)for 20 min on ice. Cells were then washed extensively to remove the freeSA-mFasL and stained with an antibody against FasL (PE-MFL4) in flowcytometry. As shown in FIG. 5, 100% of the BM cells expressed SA-mFasL.

EXAMPLE 4

Time Kinetics of Expression in Vivo

Splenocytes were labeled with a lipophilic dye (CFSE), whichincorporates and persists in the cell membrane for an extended period oftime in vivo, modified to express biotin or SA-mFasL, and injected intosyngeneic animals intravenously. Splenocytes were harvested at varioustimes post injection and stained with APC-streptavidin or anti-FasLantibody for analysis in flow cytometry. As shown in FIG. 3, biotin wasdetected on the surface of 40% of CFSE positive cells 10 days after invivo injection, the latest time point tested. Similarly, CFSE positivecells modified to express SA-mFasL were positive (26%) for theexpression of this molecule 5 days after injection. Taken together,these data clearly demonstrate that this approach allows for theexpression of proteins for an extended period of times in vivo toperform function of interest.

EXAMPLE 5

Blocking of Alloreactive Immune Response in Vitro Using Target CellsExpressing Immunomodulatory Molecules

To test whether cells modified to express immunomodulatory proteins ofinterest using this approach can be used to prevent alloreactiveresponses, allogeneic splenocytes were modified to express SA-mFasL andused as targets in in vitro proliferation assays. Briefly, splenocytesfrom F344 rats were biotinylated, treated with culture supernatants orpurified SA-mFasL. Supernatant from cells transfected with anonfunctional construct served as control and are referred to as S2supernatant throughout this application. These cells were thenirradiated and used as stimulators for alloreactive responses in astandard five-day mixed lymphocyte culture. Lymphocytes harvested fromthe lymph nodes of PVG.IU rats were used as responders. Targetsexpressing SA-mFasL completely blocked the proliferative response oflymphocytes as compared to culture without SA-mFasL. These datademonstrate that biotinylated splenocytes can serve asantigen-presenting cells and that SA-mFasL expressed on the surface ofsplenocytes block an alloreactive immune response in vitro.Additionally, these data demonstrate that biotinylation does notinterfere with the antigen-presenting function of cells since cellsdecorated with biotin and treated with S2 control supernatants did notinterfere with their capacity to stimulate lymphocytes. These data,therefore, demonstrate that expression of the proteins on the surface ofcells does not interfere with the function of cells nor the expressedmolecules. This is a critical step in validating protein-basedexpression as a means of immune regulation.

EXAMPLE 6

Toxicity of SA-mFasL Protein.

In general, most cells that express Fas are activated, such as activatedlymphocytes, or fast-dividing, such as hepatocytes, or undesirable, suchas tumor cells. However, it had to be asked whether SA-mFasL might betoxic. It has been shown that antibodies against Fas induce fulminantliver damage in selected mouse strains when injected intravenously orintraperitoneally. This is believed to be due to the expression of Fason hepatocytes. In order to ascertain whether the chimeric protein ofthis invention was likewise toxic, rats were injected intraperitoneallyor intravenously with 2×10⁷ splenocytes bearing biotin-SA-mFasL or 8×10⁷bone marrow cells bearing biotin-SA-mFasL intravenously. The animalswere closely monitored for 10 days and then sacrificed for grosspathological and anatomical analysis. No noticeable pathology was foundin animals injected with cell-biotin-SA-mFasL as shown in Table 2.

TABLE 2 Route of Group Number Cells injection Side effects I 3 2 × 10⁷splenocytes-bio intraperitoneal none II 3 2 × 10⁷ sp.-bio-SA-mFasLintraperitoneal none III 3 8 × 10⁷ bone-marrow-bio intravenous none IV 38 × 10⁷ bone-marrow-bio-SA- intravenous none mFasL

EXAMPLE 7

FasL Expressing BM Cells Rescue Lethally Irradiated Rats

The major premise of this approach is to use protein-based expression asan alternative to DNA-based gene therapy. Prevention of BM cellrejection by this approach of modifying BM cells for immune evasion willsuffice for this condition. Bone marrow cells were harvested from PVG.R8or ACI rats, modified to express biotin or SA-mFasL, and administeredi.v. at 0.7–1×10⁸ cells/animal into lethally (950 cGy) irradiated rats.Irradiated animals receiving no cells served as controls. All theanimals receiving no cells expired within 8–9 days (n=6) whereas all theanimals (n=12) receiving BM cells manipulated to express bio or SA-mFasLwere 100% chimeric (FIG. 6) and survived indefinitely (>100 days). Thesedata clearly demonstrate that BM cells are safely manipulated by thisnovel approach to express proteins of interest for therapeutic purposeswithout affecting their long-term engraftment capacity.

EXAMPLE 8

SA-mFasL Expressing BM Cells Block Alloreactive Responses in Vivo

BM cells expressing an immunomodulatory molecule such as SA-mFasL wereused to induce allotolerance as target cells in vivo. PVG.1U or WF ratswere administered i.v. with 0.7–2×10⁸PVG.R8 or ACI BM cells,respectively, expressing FasL or biotinylated cells treated with controlS2 supernatant. Mesenteric lymph nodes were harvested 60 days afterinjection and used as responders to PVG.R8 or ACI cells in a standardmixed lymphocyte assay. There was a complete absence of response todonor antigens. This in vivo immune nonresponsiveness was donor specificas the response to third party antigens was intact. This effect wasSA-mFasL-specific as animals receiving PVG.R8 cells treated with controlS2 supernatant generated a normal response to donor as well as thirdparty antigens as compared with the response of naïve animals (FIG. 7).These data validates the in vitro blocking observation and providedirect evidence for the immunomodulatory effect of this approach invivo.

EXAMPLE 9

Modification of Heart to Express CSA-Protein on Vascular Endothelium

The methods of the present invention have been applied to effectprotein-based expression on organs. Modification of organs, rather thanthe host, ex vivo to express proteins of interest presents a desiredtherapeutic approach in selected settings. Ex vivo manipulation avoidscomplications that may arise if the host were treated, and which mayinclude, but are not limited to, undesirable side effects. Therefore,the heart was used as a test system to express biotin, streptavidin, andSA-mFasL in ACI rats and B10.BR mice at 37° C. Briefly, the aortas ofexcised hearts were cannulated and perfused in a Langendorff retrogradeperfusion system at a pressure of 96 mmHg with modified Krebs-Henseleitsolution (KH). Cardiac contraction force was monitored with a latexballoon introduced into the left ventricle. The perfusion protocol forrat hearts was: 20 min with KH to allow stabilization of cardiacfunction; 20 min KH containing 5 μg/ml EZ-Link Sulfo-NHS-Biotin(Pierce); 10 min KH for biotin washout; 20 min KH containing 0.5 μg/mlof either Streptavidin-FITC (Zymed) or SA-mFasL; 10 min KH forstreptavidin washout. Control hearts were perfused with KH solutiononly. Left ventricular pressure decreased during 80 min of perfusionfrom 98±7 to 91±6 mmHg in all three groups (n=5/group). Coronary flowdid not decrease significantly from baseline values of 10.4±0.6 ml/min(n=15). These data indicate that endothelial biotinylation anddecoration with SA-mFasL have no short-term detrimental effect oncardiac function and that FasL is not directly toxic to the coronaryarteries.

It was next determined whether biotinylated endothelial lining of thecoronary arteries can be “decorated” with SA-mFasL at 4° C. Thistemperature is widely used in the clinical setting for extracorporealcardiac preservation before transplantation. Mouse (B10.BR) and rat(ACI) hearts were arrested with a magnesium cardioplegic solution (KH+16mM MgSO₄) and were perfused at 4° C. according to the protocoldelineated above (biotin, KH, streptavidin-FITC or SA-mFasL, KH). Biotinon vascular endothelium was detected by streptavidin-FITC whereasSA-mFasL was detected using antibodies against streptavidin (Zymed) andFasL (MFL4) as primary and FITC-labeled proper secondary antibodies.Hearts were removed and frozen at −80° C. on the stage of a modifiedmicrotome (Polycut S, Reichert-Jung). Sequential cryosections (˜200micron) were performed and the presence of FITC-labeled streptavidin orantibodies was determined with an Axiopan microscope (C. Zeiss). Thestage of the microscope was modified to accept the cooling block of themicrotome. Images of cardiac sections were acquired at a magnificationof 10×. Fluorescence was detected in cardiac vasculature, demonstratingthe feasibility of biotin and streptavidin conjugation to vascularendothelium under hypothermic conditions, in a preservation mediumwidely used in clinical practice. The presence of SA-mFasL in the graftendothelium perfused with SA-mFasL, but not control S2 supernatant, wasverified using FITC-MFL4 mAb. These data show that SA-mFasL can beintroduced into coronary arteries via biotinylation in conditions ofextracorporeal organ preservation. Manipulation of the graft asdescribed above is performed within 10–60 min, at 4° C., using astandard preservation solution. Thus, this approach meets therequirements of temperature, duration, and conditions used in theclinic.

EXAMPLE 10

Hearts Modified to Express Biotin or SA-mFasL Survived Indefinitely whenTransferred into Syngeneic Host.

The question arose whether this protein-based modification of organsaffects the function. Hearts modified to express bio or SA-mFasL wereheterotopically transplanted into syngeneic recipients. Graft survivalwas assessed by daily abdominal palpation of the grafted heart. As shownin Table 3, there was no detectable difference between the survival ofgraft recipients transplanted with syngeneic hearts decorated withSA-mFasL (n=4;>56 days) or KH-perfused control syngeneic hearts (n=4;>74days).

TABLE 3 Survival of heart grafts expressing SA-mFasL Group Recipient NDonor Treatment Rejection time (days) MST A BALB/c 5 BALB/c— >100, >100, >100, >100, >100 >100 B BALB/c 4 BALB/cPerfusion >74, >74, >88, >88 >74 C BALB/c 4 BALB/c Perfusion +SA- >56, >56, >68, >68 >56

EXAMPLE 11

Chimeric Decorated Islet Cells.

Pancreatic islet transplantation is widely contemplated for thecorrection of diabetes. To test whether the methods of this inventioncan be effective for the prevention of rejection of islet allografts,diabetes was induced in BALB/c recipients by intraperitoneal injectionof streptozin (260 mg/kg). Animals demonstrating blood glucoselevels>450 mg/d1 for at least three consecutive days were used asrecipients of minor+major histocompatibility antigens-disparate C57BL/10islets. Islets were harvested from C57BL/10 mice and isolated on aFicoll gradient according to standard protocol. Splenocytes wereharvested from C57BL/10, biotinylated (15 μM), decorated with FasL orcontrol supernatant, and irradiated (2000 rads). One million ofsplenocytes were cotransplanted with ˜400 islet cells under the kidneycapsule of diabetic Balb/c mice. Survival and function of the isletswere assessed by monitoring blood glucose levels starting three daysafter transplantation. FasL-decorated splenocytes prolonged the survivalof all allogeneic islets beyond nine days, the date at which the glucoselevels of control mice had risen to near the pretransplantation level.FIG. 8 shows a pancreatic cell expressing FasL and streptavidin.

In a further example of this technique, mouse pancreatic islets wereharvested by digestion with collagenase and purified using Ficollgradients. Islets were cultured overnight and then modified with biotin(5 μM) and decorated with SA-mFasL (˜100 mng/ml) or S2 supernatant ascontrol. Inspection of islets by confocal microscopy showed high levelsof biotin and SA-mFasL. Islets decorated with SA-mFasL remained viablein culture for over 10 days as determined by visual inspection andtrypan blue exclusion.

Taken together, these data clearly demonstrate that cells, tissues, andcomplex organs, such as hearts, can be modified with biotin to displayproteins chimeric with streptavidin for extended periods of time withoutdetectable toxicity.

EXAMPLE 12

Prevention of GVHD.

Bone marrow transplantation (BMT) has the potential to treat a varietyof genetically inherited and acquired hematological disorders, inducetolerance to autoimmune antigens and foreign grafts. BMT is, therefore,perceived as a natural way of performing gene therapy. The majorcomplication is, however, graft-versus-host disease, which is fatal inmost instances. T cells are considered to be the most important cells tocause GVHD. A method allowing specific elimination of T cells in the BMinoculum that cause GVHD has wide-spread clinical application.Conventional gene therapy has been used to introduce “suicide” genesinto T cells. Once GVHD occurred, the suicide machinery was activated toeliminate these cells. The obstacles of conventional gene therapy,however, are major problems for routine use of this approach in clinics.A more “noninvasive” approach is needed to achieve this “suicide”approach.

This methods of the present invention are well suited for the “suicide”approach or immune down-regulation. Display of apoptotic (death ligands)or down-regulatory molecules (anti-inflammatory cytokines; CSA-IL-4,IL-10, TGF-β) in bone marrow cells (including mature T cells) areanticipated to physically or functionally eliminate these cells upontheir recognition of host antigens and activation. As a proof ofconcept, it was shown that T cells in splenocytes modified to displaySA-mFasL did not generate a proliferative response when stimulated withT-cell mitogens (FIG. 10). In contrast, unmodified splenocytes generateda vigorous proliferative response, demonstrating that T cells respondingto mitogens expressed Fas and died upon the interaction of FasL with Fason the same cell (suicide). It was further demonstrated that ratsreceiving bone marrow cells and mature lymphocytes developed GVHD within17–20 days. Treatment of these animals with syngeneic splenocytesexpressing FasL resulted in the prevention of GVHD, strong in vivo datathat this approach is superior to other treatment regimens that are usedin the clinic.

EXAMPLE 13

Immune Down-Regulation Using Immunomodulatory Molecules.

The methods of the present invention of expressing exogenous proteins onthe cells, tissues, and organs are made particularly effective indown-regulating the immune system for therapeutic purposes when severalof these molecules are expressed simultaneously. Therefore, acombination of death ligands (SA-mFasL, -TNFα, -TRAIL and likes) andanti-inflammatory molecules (CSA-IL-4, IL-10, -TGF-β and likes) areexpressed on the surface of cells, tissues, and organs as a therapeuticapproach to prevent/treat autoimmune disease, foreign graft rejection,and other immune-based disorders. This method shows down-regulation ofthe immune system.

EXAMPLE 14

Vaccination Against Tumors.

Tumors may evade the immune system by down-regulating the signals thatprovoke a T-cell response. An effective T-cell response requires threedistinct signals: Signal 1, 2, and 3. Signal 1 is generated by T-cellreceptor interaction with the major histocompatiblity complex (MHC)anti-peptide on antigen-presenting cells (APCs). Signal 2 is mediated bythe engagements of costimulatory molecules, such as B7/CD28 andCD40/CD40L, on T cells and APCs. Signal 3 is transduced via cytokineselaborated by T cells and APCs that have received both Signal 1 and 2.The transduction of these three signals drives T cells and APCs toproliferation and differentiation into effectors for the generation of aproductive immune response. The lack of any of these signals during Tcell response to tumors may serve as one the most effective mechanismsby which tumors evade the immune system. The present invention providesmethods and compositions to modify cell surface for the expression ofexogenous proteins. This approach is aimed at converting any cells intoprofessional antigen-presenting cells for the generation of an effectiveimmune response.

A chimeric protein composed of CSA and extracellular portion of CD40Lwith engineered synthetic residues for structural flexibility(CSA-CD40L) was produced in Drosophila production cells. This moleculewas then used for expression on the surface of splenocytes. Almost allthe splenocytes were positive for CSA-CD40L as shown by an antibodyagainst CD40L in flow cytometry (FIG. 11). Splenocytes expressing CD40Lwere much more effective than regular splenocytes at inducingantigen-specific responses when used as APCs. This clearly demonstratesthe feasibility of our approach to express exogenous proteins on thecell surface for therapeutic purposes.

This concept is tested in a lung carcinoma animal model. Two lungcarcinoma lines of mouse origin, Lewis and Line 1 and the C58 tumorcells (rat lymphoma) will be used for the purpose of vaccination againsttumors.

Chimeric proteins produced from nucleic acids comprising the nucleicacids of CSA fused to the nucleic acids of B7.1 (SEQ. ID No. 2), CD40L(SEQ. ID No. 6), or IL2 (SEQ. ID No. 4) will be used for display on thesurface of these tumor cells and the cells will be used for vaccinationof mice and rats against tumors. Briefly, tumor cells (2×10⁷) will bebiotinylated as above. After several washes with PBS, the biotinylatedcells will be incubated with chimeric proteins as above to bind thechimeric protein to the biotinylated cells. The chimera-decorated cellswill be irradiated at 5–10,000 cGy and injected into tumor bearinganimals to serve as a vaccine. Tumor regression is monitored. Based onthe results as shown in the above examples, it is expected that thismethod will show up-regulation of the immune system.

It can be easily seen that other tumor systems and chimeric proteins canbe identified, and the methods of this invention applied to provideother tumor vaccines and vaccines against infections.

This invention has been described in various preferred embodiments.Those skilled in the art will readily recognize that modifications,deviations or substitutions of the compounds or methods here disclosedmay be made without departing from the spirit and scope of thisinvention. All such modifications, deviations and substitutions areconsidered to be within the scope of the claims of this invention.

1. A protein encoded by a nucleic acid having the sequence of SEQ IDNO:2.
 2. A nucleic acid having the sequence of SEQ ID NO:2.
 3. Anisolated cell comprising a protein encoded by a nucleic acid having thesequence of SEQ ID NO:2.
 4. The isolated cell of claim 3, wherein thecell is selected from the group consisting of splenocytes, tumor cells,bone marrow cells, endothelial cells, and islet cells.
 5. An isolatedproduction cell comprising a nucleic acid having the sequence of SEQ IDNO:2.
 6. A polypeptide product produced by the production cell of claim5, wherein the polypeptide product is encoded by the nucleic acid havingthe sequence of SEQ ID NO:2.
 7. An isolated target cell comprising aprotein encoded by a nucleic acid having the sequence of SEQ ID NO:2,wherein the protein is displayed on the surface of the target cell. 8.The isolated target cell of claim 7, wherein the cell is selected fromthe group consisting of splenocytes, tumor cells, bone marrow cells,endothelial cells, and islet cells.
 9. A tumor cell comprising a proteinencoded by a nucleic acid having the sequence of SEQ ID NO:2, whereinthe protein is displayed on the surface of the tumor cell.
 10. Avaccine, wherein the vaccine comprises a number of tumor cells decoratedwith protein encoded by a nucleic acid having the sequence of SEQ IDNO:2 sufficient to generate a T-cell response when administered to atumor-bearing animal.
 11. A method of attaching an immune costimulatoryagent to a tumor cell surface in vivo, the method comprising: contactinga biotinylated tumor cell with a chimeric protein comprising (i) theimmune costimulatory agent and (ii) core streptavidin, wherein theimmune costimulatory agent comprises the extracellular domain of B7.1,to form a tumor cell having a surface decorated with the chimericprotein.
 12. The method of claim 11, wherein the tumor cell is a targetcell.
 13. The method of claim 11, wherein the chimeric protein isencoded by a nucleic acid having the sequence of SEQ ID NO:2.
 14. Themethod of claim 11, wherein the method comprises administering thechimeric protein to a subject containing the biotinylated tumor cell.15. The method of claim 11, further comprising the step of biotinylatinga tumor cell to form the biotinylated tumor cell.
 16. The method ofclaim 15, wherein the tumor cell is biotinylated at room temperature.17. The method of claim 15, wherein the biotinylating step comprisescontacting the tumor cell with a biotin-containing compound for a periodlasting no more than about 30 minutes.
 18. The method of claim 17,further comprising the step of washing the tumor cell to remove freebiotin-containing compound.
 19. The method of claim 17, wherein thebiotin-containing compound comprises Sulfo-NHS-LC-biotin.
 20. The methodof claim 15, wherein the biotinylating is effected in vivo.
 21. Themethod of claim 20, wherein the method comprises administering abiotin-containing compound to a subject containing the tumor cell. 22.The method of claim 21, wherein the biotin-containing compound comprisesSulfo-NHS-LC-biotin.
 23. The method of claim 15, wherein thebiotinylating is effected in vitro.